Progression of cells from G2 phase of the cell cycle to mitosis is a tightly regulated process that requires activation of the Cdc2 kinase, which determines onset of mitosis in all eukaryotic cells. In fission yeast (Schizosaccharomyces pombe), the activity of Cdc2 is regulated by the phosphorylation status of tyrosine 15 (Tyr 15) on Cdc2, which is phosphorylated by Wee1 and Mik1 tyrosine kinases during late G2 and is rapidly dephosphorylated by the Cdc25 phosphatase to trigger entry into mitosis. Cdc25, Wee1 and Mik1 are the targets of two well-characterized regulatory pathways, the DNA damage and DNA replication checkpoints, which both cause cell cycle arrest by inhibitory phosphorylation of Tyr15 on Cdc2 primarily through inhibition of Cdc25.The viral protein R (Vpr) of HIV-1 induces G2 arrest in cells from distantly related eukaryotes, including fission yeast and human. Similar to the two checkpoint pathways, Vpr also induces G2 arrest in fission yeast through inhibitory phosphorylation of Tyr15. However, Vpr does not use any of the early or late steps in the checkpoint pathways, and instead it acts primarily through activation of Wee1, protein phosphatase 2A (PP2A) and to a lesser extent inhibition of Cdc25, suggesting that Vpr induces G2 arrest through a novel PP2A-mediated regulatory pathway. The goal of this proposal is to use fission yeast as a model system to test this hypothesis and to elucidate this new G2/M regulatory pathway. The three specific aims are to 1) define the regulatory effects of PP2A on Wee1 and Cdc25 when Vpr induces G2 arrest; 2) characterize a potential protein complex of Vpr with PP2A and casein kinase II alpha (CKIIa) and define the role of this complex in Vpr-induced G2 arrest, and 3) characterize four multicopy Vpr-specific suppressors (Wos2 and Vsp24C8)/enhancers (Rad25 and Sum1) and their role in Vprinduced G2 arrest. These proposed studies will provide a comprehensive framework for this new cell cycle control pathway and provide insights into fundamental aspects of this new G2/M surveillance system of eukaryotic cells.